One of the more common neurologic diseases in human adults is multiple sclerosis. This condition is a chronic, inflammatory CNS disease characterized pathologically by demyelination in the brain and spinal cord. Glatiramer acetate (GA), also known as Copolymer-1, has been shown to be effective in treating multiple sclerosis (MS). Daily subcutaneous injections of Glatiramer acetate (20 mg/injection) reduces lesions, relapse rates and progression of disability (Johnson K. P. et al., Neurol., 1995, 45(7): 1268-76). Glatiramer acetate reduces the proportion of new MS lesions evolving into “black holes” (Filippi M. et al., Neurol., 2001, 57:731-733).
Glatiramer acetate (Copolymer-1) is marketed under the brand name COPAXONE®. It is approved for reducing the frequency of relapses in patients with Relapsing-Remitting Multiple Sclerosis (RRMS). Glatiramer acetate consists of the acetate salts of synthetic polypeptides, containing four naturally occurring amino acids: L-glutamic acid, L-alanine, L-tyrosine and L-lysine with an average molar fraction of 0.141, 0.427, 0.095 and 0.338 respectively. It is synthesized by chemically polymerizing the four amino acids to yield the product with the desired molecular weight range. The average molecular weight of Glatiramer acetate is 4,700-11,000 daltons [US label for lyophilized powder] or 5,000-9,000 daltons [US label for pre-filled syringe/Summary of Product Characteristics]. Copaxone comprises a mixture of polypeptides having different molecular weights and sequences.
The structural formula of Copaxone is(Glu,Ala,Lys,Tyr)x.xCH3COOH(C5H9NO4.C3H7NO2.C6H14N2O2.C9H11NO3)x.xC2H4O2 
The process for preparation of Glatiramer acetate is described in Euro. J. Immun. 1, 242-248 (1971) [Tietelbaum et al] and U.S. Pat. No. 3,849,550 [Tietelbaum et al]. U.S. Pat. No. 3,849,550 discloses a process, wherein the N-carboxyanhydrides of tyrosine, alanine, γ-benzyl glutamate and ε-N-trifluoroacetyl lysine are polymerized at ambient temperature in anhydrous dioxane with diethylamine as initiator. The deblocking of the γ-carboxyl group of the glutamic acid is effected by hydrogen bromide in glacial acetic acid which is followed by the removal of the trifluoroacetyl groups from the lysine residues by 1M piperidine.
Process for preparation of copolymer-1 is also disclosed in U.S. Pat. No. 5,800,808, IN190759, U.S. Pat. No. 5,981,589, U.S. Pat. No. 6,054,430, U.S. Pat. No. 6,342,476, U.S. Pat. No. 6,362,161 and WO00/05250. These documents elaborate on the process for preparing copolymer-1 involving polymerization of four N-carboxyanhydrides of amino acids to obtain protected copolymer and deprotection of the protected copolymer using hydrogen bromide in acetic acid and piperidine to obtain deprotected copolymer which is then subjected to dialysis for purification and salt exchange.

US20060172942 discloses process for preparation of Glatiramer acetate which involves polymerizing N-carboxyanhydrides of tyrosine, alanine, γ-benzyl glutamate and trifluoroacetyl lysine to obtain protected polypeptides. The benzyl protecting group from the protected polypeptides is removed by catalytic hydrogenolysis followed by removal of the trifluoroacetyl protecting group by contacting the polypeptide with an organic base. The free trifluoroacetyl groups and low molecular weight impurities are removed from the polypeptide mixture by ultrafiltration followed by contacting the polypeptide mixture with an aqueous solution of acetic acid to obtain the acetate salt of polypeptide.
U.S. Pat. No. 7,049,399 describes a process for the preparation of a polypeptide comprising polymerization of a mixture of the N-carboxyanhydrides of L-alanine, L-tyrosine, a protected L-glutamate and a protected L-lysine to obtain protected copolymer or salt thereof; deprotection of the protected copolymer (or salt thereof) to produce polypeptide or a pharmaceutically acceptable salt thereof in one single step; separation and purification of the polypeptide (or a pharmaceutically acceptable salt) to obtain a purified polypeptide. The deprotection of the protected copolymer is carried out either by catalytic transfer hydrogenation or catalytic hydrogenation under hydrogen pressure ranging from 40-100 psi and at temperature ranging from 50°-80° C.
The disadvantage with respect to the process disclosed in these documents is that hydrogenolysis requires high pressure and temperature, which in turn needs additional operation precaution on large scale, thus increasing the production cost. Since Copaxone is stable at a low temperature of 2° C. to 8° C., high temperature during final step may result in degradation of the product thereby reducing the quality and yield of the desired product.
WO2006050122 describes a two step process for preparing Glatiramer acetate. The process involves polymerizing a mixture of N-carboxyanhydride of L-tyrosine, N-carboxyanhydride of L-alanine, N-carboxyanhydride of protected L-glutamate and N-carboxyanhydride of N-protected L-lysine, in a polar aprotic solvent in presence of an initiator to form a protected Glatiramer; adding an acid and/or organic or inorganic base to the formed protected Glatiramer to form Glatiramer; and treating the obtained Glatiramer with acetic acid to form Glatiramer acetate. The deprotection of the protected Glatiramer is carried out by two different methods. First method for deprotection is by treatment with an acid and second method for deprotection is accomplished by treatment with alkaline earth metal hydroxide. The disadvantage of this process is that use of alkaline earth metal hydroxide for deprotection of peptides result in slow reactions and formation of high levels of the diastereomer resulting from racemization/epimerization of the stereogenic centers (Ahmed F. Abdel-Magid et. al., Tetrahedron Letters 39, 3391 (1998)).
WO2006029393 discloses a process of producing a mixture of trifluoroacetyl polypeptides which do not all have the same amino acid sequence, where each polypeptide consists essentially of alanine, glutamic acid, tyrosine and lysine, wherein the mixture has a desired average molecular weight comprising deprotecting a mixture of polypeptides each consisting essentially of alanine, γ-benzyl glutamate, tyrosine and trifluoroacetyl lysine with a solution of hydrobromic acid in acetic acid, which solution comprises less than 0.5% of free bromine and less than 1000 ppm of metal ion impurities. It further discloses that solution of hydrobromic acid in acetic acid having less than 0.5% of free bromine can be achieved by pretreating the solution with a bromine scavenger, preferably phenol to decrease the level of free bromine. It also discloses the use of 10% to 36% of hydrobromic acid in acetic acid.
US20080021200 discloses a process for preparing a polypeptide comprising L-tyrosine, L-alanine, L-glutamate and L-lysine, or a pharmaceutically acceptable salt thereof, wherein said process comprises, (a) polymerizing a mixture of N-carboxyanhydride of L-tyrosine, N-carboxyanhydride of L-alanine, N-carboxyanhydride of a protected L-glutamate and N-carboxyanhydride of a protected L-lysine, in a polar aprotic solvent in the presence of an initiator, to form a protected polypeptide; (b) admixing an acid with the protected polypeptide formed in Step (a) and a solvent, to form a product; and (c) admixing a substance selected from the group consisting of diisopropylamine, isopropylamine, ammonia, and mixtures thereof, with the product formed in Step (b), and water or a mixture of a solvent and water, to form a deprotected polypeptide or a pharmaceutically acceptable salt thereof. It further discloses that diisopropylamine and isopropylamine were the only amines that successfully removed the Nε-trifluoroacetyl group of lysine moiety. It also discloses that when diisopropylamine or isopropylamine is used for removal of trifluoroacetyl groups, a clear solution is obtained after about 1 hr or after about 1.5 hrs respectively.
WO2009016643 describes a process for preparation of Glatiramer acetate (Copolymer-1) where the trifluoroacetyl copolymer-1, obtained after the debenzylation reaction, is washed with an organic solvent to remove reactive benzyl bromide, generated as a reaction by-product. The released benzyl bromide is a highly reactive electrophile and reacts with nucleophiles like primary and secondary amines to generate unwanted N-alkylated products. Also it is highly lachrymatory and handling it in large quantities on commercial scale is hazardous and unsafe. WO'643 also discloses debenzylation reaction of protected copolymer-1 for shorter duration of time at a higher temperature and a method to remove benzyl bromide from the reaction mixture. Removal of benzyl protecting group of glutamic acid is affected by 33% hydrogen bromide in acetic acid at 35-45° C. for 1-5 hours thereby reducing the reaction time, and in order to eliminate benzyl bromide the filtered product is washed with an organic solvent. This method employs an additional organic solvent which is undesirable on a commercial scale. Thus the processes for preparation of Glatiramer acetate as disclosed above are industrially not feasible.
The inventors of the present invention have developed a simple, cost effective, efficient, commercially viable and consistent process for preparation of copolymer-1 (Glatiramer acetate). Use of 33% HBr leads to cleavage of the peptide bonds in the polymer resulting in low molecular weight compounds. The present invention provides a more commercially viable process for preparation of Glatiramer acetate by using about 7% to 20% HBr, preferably about 15% HBr in acetic acid for removal of the benzyl protecting group at room temperature, preferably at about 23° C. to 30° C., more preferably at about 25° C., most preferably at 25° C.±0.2° C. The present invention further provides a process for preparation of Glatiramer acetate where the wet solid obtained after the first deprotection step is not subjected to drying and is used as such for the further reaction which saves considerable time and hence is considered to be an industrially viable process. The present invention further provides a process for removal of the trifluoroacetyl protecting group using amine, preferably secondary amine such as dialkyl amine selected from diethylamine, dimethyl amine or diisopropylamine, preferably diethyl amine.
Prior art provides methods for determination of molecular weight of Polypeptides, in particular Glatiramer acetate. WO200018794 (WO'794) discloses use of a plurality of molecular weight markers to establish a relationship between retention time on a chromatographic column and molecular weight for determination of average molecular weight of glatiramer acetate batches. Each of these molecular weight markers disclosed in WO'794 is a polypeptide and has a pre-determined amino acid sequence and defined molecular weight. The present invention provides the use of polypeptide fractions (molecular weight markers), obtained by subjecting Copaxone/Glatiramer acetate/Glatiramoids to Size Exclusion Chromatography (SEC) or Gel Permeation Chromatography (GPC), for determination of molecular weight of Glatiramer acetate batches. The polypeptide fractions (molecular weight markers) obtained by the process of the present invention has a defined molecular weight. However, the amino acid sequence of these polypeptide fractions are random.
Another analytical technique used for characterizing a polypeptide/protein is Peptide Mapping. Peptide mapping is a comparative procedure where the information obtained, compared to a reference standard or reference material similarly treated, confirms the primary structure of the polypeptide/protein. This helps in detecting whether alterations in structure have occurred and demonstrates process consistency and genetic stability. WO2010129851 provides a process whereby hydrolysis enzymes are used to digest a standard of a complex mixture of polypeptides such as Glatiramer acetate, into several peptide fragments. The peptide fragments are analyzed by mass spectrometry. The mass spectrometric results of each sample are used as the fingerprint for comparison with other samples. Each peptide fragment detected by the first mass analyzer is selected and subjected to second mass spectometric analysis to cleave the precursor peptide ions into even smaller fragments. The mass spectra obtained from MS/MS analysis are analyzed by the software such as Biotools to obtain the sequence of each peptide fragment.
Expert Opin. Pharmacother. (2009) 10(4) discloses that polypeptide mapping using capillary electrophoresis separation of polypeptide fragments obtained after digestion with trypsin and mapping based on proteolytic hydrolysis by carboxypeptidase P followed by separation of the fragments by reverse-phase HPLC are methods of discerning sequence differences among GA structures and those of other glatiramoids. Carboxypeptidase P is an expensive reagent and not easily available. The inventors of the present invention tried various combinations of proteolytic enzymes. It was found by the inventors of the present invention that a mixture of polypeptides treated with trypsin followed by treatment with carboxypeptidase B provides results similar to that of carboxypeptidase P.